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FOCiS Advanced Course in Basic & Clinical Immunology
Innate Lymphocytesand iNKT cells
Lewis L. [email protected]
iNKT cell Innate lymphocytes
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ILC3
NKR+ ILC, LTi
Common Lymphoid Progenitor
NK cell
ILC2
Id2Ets1Nfli3
Zbtb16Gata3
IL-15
ILCProgenitor
RORα
RORγt
IL-7
IL-7
ILC1 IFNγ TRAIL
IFNγperforin
IL-17IL-22
IL-5IL-13
Eomes
Tbet
Spits & Cupedo Ann Rev Imm 2012
ILC2 –protect against parasites and help wound repair
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Spits & Cupedo Ann Rev Imm 2012
ILC3 –protect against extracellular bacteria and help wound repair
IL-17
Lim et al. Curr Op Imm 2017
Innate Lymphoid Cells adapt to their environment
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Natural Killer versus “Natural Killer T” (NKT) cells
What is the difference?NKT cells are T cells!
rearrange TcR genes, express an invariant αβ-TcR, require a thymus for development
NK cells are not T cells!
don’t rearrange TcR genes or express CD3 on the cell surface
do not require a thymus
Primordial MHC
MHC I MHC IICD1
MHC and CD1 Evolution and Function
Tim
e
Present
Peptides Lipids Peptides
Thanks Mike Brenner
CD8+ T cell CD4+ T cellNKT
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Lipid in CD1d binding groove
Top View
Side View
Thanks Mike Brenner
Defining iNKT cells
T cells expressing an invariant TcRα chainand recognizing lipid antigens presented by CD1d
In humans - invariant Vα24 + Jα18 TcRα
In mice - invariant Vα14 + Jα18 TcRα
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Frequency and distribution of iNKT cells
In mice - <0.5% in blood & peripheral lymph nodes (LN)- 0.5-1% in thymus, spleen, mesenteric LN
- ~20-30% of T cells in liver- CD4+ and CD4-,CD8- subsets
In humans-Typically 10- to 100-fold less abundant-CD4+, CD8+, and CD4-,CD8- subsets
–not prevalent in liver
iNKT cells may play an important role in control of many infections
Thanks Mike Brenner
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Cohen, Garg, and Brenner Adv Imm 2009
How do iNKT cells get activated?
Microbial lipid antigens
Jα28-/-
wt
CFU lung day 3 p.i.
Kawakami EJI 33:3322, 2003
iNKT cells protect mice against Streptococcus pneumoniae
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Few lipid antigens found that activate iNKT cells
Mic
robi
al li
pid
antig
ens
for
iNK
TaGalCer - Synthetic
BbGL-IIc - Borellia burgdorferi
a-glucosyldiacyglyerol- Strept pneumoniae
Brigl et al. Nat. Immunol. 2003
iNKT cells are strongly stimulated by CD1d+ dendritic cells pulsed with many types of bacteria
DCiNKT
Gram +Gram -
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iNKT Cells w/o DC
Brigl et al. Nat. Immun. 2003
IL-12 and dendritic cells induce iNKT cell activation
(without foreign antigens, but requires CD1d + self-lipids)
IL-12 ng/ml
DC
iNKT
Self-lipids recognized by iNKT- iGB3- plasmalogen lyso PE- aGlcDAG (vaccenic acid)
Direct & indirect activation of iNKT cells
Cohen, Garg, and Brenner Adv Imm 2009
Selected microbesS. pneumoniae – aGlcDAGBorellia – a-GalDAGSphingomonas – a-GluCer
Most microbesTLR ligands induce IL-12+CD1d self-lipids
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Subsets of iNKT cells specialize in cytokine production- similar to Th and ILC subsets
Prevention of autoimmune diseasesdiabetes, collagen-induced arthritis, EAE
Autoimmune diseasesairway hyper reactivitycontact hypersensitivityasthma
iNKTCells
IL-4IL-13
Inflammatory/autoimmune diseasesNZB/NZW lupusgraft vs. host diseaseatherosclerosis
Fight infections & cancerbacterial, parasitic, viral and tumor rejection
Exacerbates
Ameliorates
IFNγIL-17
IFNγIL-4 IL-13
NKT cells function in a variety of immune responses via secretion of Th1-, Th2-, or Th17-type cytokines
Thanks Mitch Kronenberg
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Questions about iNKT cells?
3rd lineage of lymphocytes
Function in innate immunity to protect against viruses, bacteria, parasites, fungi, & tumors
Produce cytokines & kill abnormal cells
Human CD3-,CD56+, (Nkp46+)Mouse CD3-,NKR-P1C+ (aka NK1.1) (NKp46+)
Natural Killer Cells
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Detecting NK cells in human peripheral bloodC
D56
CD3
CD56dim mature CD56bright immature
New England Journal of Medicine 1989
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Physiological role of NK cellsis to protect against viral infections and cancer
Humans lacking NK cells are particularly susceptible to:
- Epstein-Barr Virus Fleisher, J. Pediatrics 1982
- Cytomegalovirus and other herpesviruses Biron, NEJM 1989
- Papillomavirus (cervical cancer) and Herpes Simplex Virus Ballas, J. Allergy Clinical Immuno 1991
- Varicela Zoster Virus Etzioni, J. Peditrics 2005
NK cell-deficient patient – caused by loss of one good GATA2 gene
CD3-CD56+ NK cells
CD3+CD56+ T cells
caused by heterozygous loss of GATA2
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NK cells are critical to early innate host defense against pathogens
0
20
40
60
80
100
% S
urvi
val
0 5 10 15Day
WT B6 Littermates
NK-Deficient B6 mice
Mouse Cytomegalovirus105 pfu i.p.
Loh JVI 2005
Human NK Cells - Where do they live?
~5-20% peripheral blood lymphocytes
~5% lymphocytes in spleen, abundant in liver
Low frequency in thymus, bone marrow, uninfected lymph nodes and lymphatics – but increase with infection
>70% of lymphocytes in decidual tissue
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NK Cells - What do they do?
Cell mediated-cytotoxicity – natural killing
Antibody-dependent cellular cytotoxicity (kill antibody-coated cells via activating Fc receptor CD16)
Early γ-interferon production
Secrete TNFα, LTα, GM-CSF, IL-3, M-CSF, IL-10, MIP-1α, MIP-1β, RANTES, etc.
(but NOT IL-2, IL-4, IL-17, or IL-22)
IL-15IL-12
IFN-γpDC
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How do NK cells sense their environment?
Cytokines produced by infected epithelial or myeloid cells
Epithelial or Myeloid cell
TLR
Bacteria, virus, parasites,fungus
Cytokines (IL-15, IL-12, IL-18)
NK cell Cytokine receptor
Interferon-γChemokines
eron-γ
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“Stressed” cells – upregulate host-encoded ligands for activating NK receptors
Host cell Host-encoded
“stress-induced” protein
Bacteria, virus, fungus, parasite
NK cell
“Stress-induced ligand” receptor
(e.g. NKG2D)
Kill!Interferon-γ
DNA damage(tumors)
Host cell “
Infected cells express virus-encoded ligands for activating NK receptors
Host cell
Virus-encodedprotein
NK cellVirus-specificNK receptor
Kill!Interferon-γ
Host cell VV
Viral infection
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How are NK cell responses regulated?
Class I+ tumorsgrow in vivo
Class I- tumorsare rejected
Class I- tumorsin NK-depleted
mice grow in vivo
NK Cells Reject Tumors Lacking MHC Class I
NK cells like to kill cells lacking MHC class I – missing-self
Karre et al. 1986 Nature 319:675
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Physiological role for NK cell inhibitory receptors for MHC class I- Detection of virus-infected cells?
Virus Protein Effect on class IAdenovirus E3-k19 Retain in ERHSV-1,2 ICP47 Blocks TAPEBV EBNA1 Block peptide generationHCMV US2, US11 ER to cytosolHCMV US3 Retain in ERHCMV US6 Blocks TAPHCMV US10 Degrades HLA-GMCMV m152 Retain in ERMCMV m04 Associates with H-2MCMV m06 Lysosomal degradationHHV8 K3, K5 Endocytosis HIV-1 Nef Endocytosis
Structural differences betweenMHC class I-specific inhibitory receptors
in mice and humans
s-s
N N
MouseLy49
HumanKiller cell Ig-like Receptor (KIR)
C
ITIM
2 (or 3) Immunoglobulin -like domains
Long cytoplasmic domain
Type II Type I
C-typelectin-like
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KIR2DL1 : HLA-C2 allotypes (Cw2, 4, 5, 6=Lys80)
KIR2DL2 & KIR2DL3 : HLA-C1 allotypes
(Cw1, 3, 7, 8=Asn80) KIR3DL1 : HLA-Bw4 KIR3DL2: HLA-A3
HLA specificities of human KIRs
C
KIR2DL
C
KIR3DL
Lanier, Ann Rev Imm 2005
2DL22DS2 2DL5B 2DS3 2DL1 3DP1 2DL5A 2DS4 2DS5 2DS1 3DL2
3DL1
2DL42DP12DL33DL3
A haplotype
2DL1 2DS4 3DL23DL12DL42DL33DL3
B haplotype
2DL22DS2 2DL1 2DL5A 2DS1 3DL23DS12DL43DL3
3DS1
2DS5
KIR genes are highly polymorphic!
Chromosome 19q13.4
2DL22DS2 2DL5B 2DS3 2DL1 3DP1 2DL5A 2DS4 2DS5 2DS1 3DL2
3DL1
2DL42DP12DL33DL3
A haplotype
2DL1 2DS4 3DL23DL12DL42DL33DL3
B haplotype
3DS1
Chromosome 19q13.4
907 alleles at 14 KIR loci
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Killer Cell Ig-like Receptors (KIR)
Ig superfamily
7-12 functional genes on human chromosome 19q
Extensive allelic polymorphism (907 alleles!) no rearrangement; mono-allelic expression possible
Expressed by subsets of NK cells and memory T cells (mostly CD8+ T cells, but also CD4+ T cells)
Inhibitory KIR2DL recognize HLA-C; KIR3DL recognize HLA-A, -B
Activating KIR - no intrinsic signaling -associate with DAP12 ITAM-adapter protein
“Missing-self” MHC on a cell is not sufficient for an NK cell to attack
NK cells require activating receptors to detect ligands on the target cell to
initiate a response
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You may have more than 30,000 NK cells subsets in your blood- CyTOF analysis by Catherine Blish (Stanford)
Ljunggren & Malmberg NRI 2007
Factors boosting NK cell lytic activity
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Koch Trends Imm 2013
Natural Cytotoxicity Receptors
ITAM-coupled activating receptor
NKp46 expressed by most NK cells in humans and mice
NKp46 also expressed by some ILC and γδ T cells
NKp30 & NKp44 in humans, not mice
Involved in recognition and killing of certain tumors
Ligands poorly defined – broadly distributed (except B7-H6 for NKp30)
NKG2D– C-type lectin-like superfamily– 1 gene, non-polymorphic, conserved mice - humans– Homodimer expressed on all NK cells, γδ T cells, and CD8+ T cells– R in transmembrane associates with D in DAP10 transmembrane
DAP10– 10 kd homodimer– Cytoplasmic YINM recruits Grb2 & p85 PI3-kinase
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H60c H60b6H6H60c0c0c0c 6H66H666H666H6H6H6666H6H6H6H6H66H66H66H66666H666H6H6H66H666H66H66H6H6666H666H6H66H666H666H66H6666666H666H66H6H666H6H6H6H6H6H6HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH 0bbbbbbbbb0bbbbbbbbbbbb0b0b0b0b0b00000
Lanier Cancer Immunology Research 2015
NKG2D has many polymorphic ligands in humans and mice
107alleles
42alleles
NKG2D ligands• MHC class I-like
– don’t require peptide or b2-microglobulin
• Bind with nM affinity to NKG2D
• Low levels expressed on healthy tissues
• Induced on virus-infected cells and tumor cells
• Induced by DNA damage (ATM/ATR pathway)
• Elevated in autoimmune diseases • (rheumatoid arthritis, celiac disease, diabetes, atherosclerosis)*
*Disclosure – I have licensed patents on blocking NKG2D in autoimmune disease
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NKG2D on NK cells, γδ T cells and CD8+ T cellsdetect NKG2D ligands on abnormal cells
Therapeutic Uses of Natural Killer Cells
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NK cell Cellule normale
+
Lysis
+
+
CD16 Tumor antigenAntibodies
Tumor cell
-
Inhibitory receptor MHC class I
Antibody-dependent cellular cytotoxicity
Rituxan, Herceptin, Erbitux, Daratumumab
NK cell Cellule normale
+
Lysis
+
+
Activating receptors
Tumor antigenBispecific antibodies
Tumor cell-
Inhibitory receptor MHC class I
Bispecific antibodies
– anti-tumor x anti-NK activating receptor
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Tumor cell
LysisInhibitory receptor
Activating receptor
Inhibitory ligand
Stimulatory ligand
Checkpoint blockade therapies
NK cell
+
-
anti-KIR
anti-NKG2A
anti-PD1
anti-Tim3
anti-LAG3
Therapies that up-regulate stress-induced ligands on tumors
or agents that activate NK cells
NK cell
+
-
Lysis
Stressed or damaged cell
Inhibitory receptor MHC class I
+
+
Activating receptors
Stimulatory ligands
Irradiation
Chemotherapy
Interferon-α/β
IL-15
IL-12
Agonist costimulatory antibodies
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NK cell
+
Lysis
+
+
CARanti-CD19-cytoplamic CD137-CD3ζ
Tumor antigen
Tumor-
Inhibitory receptor
MHC class I
CAR NK cells
Chimeric antigen receptors
Guerra Immunity 2008
Increased incidence of myc oncogene-induced B cell lymphomas in NKG2D-deficient mice
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NATURAL KILLER CELLS REMEMBER
Cytomegalovirus-seropositive humans have a unique population of CD57+NKG2C++ NK cells
0 103 104 105<PE-Cy5-A>: CD57
0
103
104
105
<PE-
A>: N
KG2C
0.62
2.77 2.44
6133.8
HCMV-seronegative
NK
G2C
HCMV-seropositive
0 103 104 105<PE-Cy5-A>: CD57
0
103
104
105
<PE-
A>: N
KG2C
43.1
7.27 45.5
13.833.4
CD57
CD57+NKG2C++
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NK cells expressing the activating CD94-NKG2C receptor are expanded in healthy humans infected with CMV
CMVneg CMV+
0.0
2.5
5.0
**
5
15
25
50p<0.001
Patient 4
0 2 4 6 8 10 12 14 16 181.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
CD57+NKG2Chi NK
HCMV DNA
0
25000
50000
75000
Time (days)
Patient 3
0 25 50 75 100 1250
2
4
6
8
CD57+NKG2Chi NKHCMV DNA
1024204840968192163843276865536131072262144
Time (days)
Patient 1
0 5 10 15 20 250
2
4
6
8
10
12
CD57+NKG2Chi NK
1000
10000
100000
1000000
HCMV DNA
Time (days)
Patient 2
0 5 10 15 20 25 30 350
2
4
6
8
10
CD57+NKG2Chi NK
HCMV DNA
0
10000
20000
30000
Time (days)
Viral load
NKG2C++ NK cells
Antiviral drugtherapy
NKG2C++ NK cells specifically expands after CMV reactivation in immunosuppressed solid organ transplant patients
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ILC and NK cells
• ILC – family of innate lymphocytes – rapid cytokine production
• NK cells keep you alive during certain viral infections
• NK cells regulated by inhibitory and activating receptors
• NK receptors are evolving rapidly
• NK cells possess immunological memory
Recent Reviews
ILC1 and NK Spits et al. Nature Immunology17:758, 2016
NK Cerwenka & Lanier Nature Review Immunology 16:112, 2016
ILCLim et al. Curr Opin Immunol 44:61, 2017Klose & Artis Nature Immunology 17:765, 2016
iNKT cellsCrosby & Kronenberg Immunogenetics 68:639, 2016
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